KR100374448B1 - DNA encoding enzymes, recombinant DNA (DNA) and enzymes, transformants and methods of making them - Google Patents

Abstract

A DNA encoding an enzyme, which forms non-reducing saccharides having trehalose structure as an end unit from amylaceous saccharides having a degree of glucose polymerization of 3 or higher, enables an industrial-scale production of a recombinant enzyme with such enzyme activity. Non-reducing saccharides obtainable by the recombinant enzyme can be used in a variety of food products, cosmetics, pharmaceuticals and feeds because of their substantial non-reducibility, mild and high-quality sweetness, adequate viscosity, and moisture-retaining ability.

Description

DNAs encoding enzymes, recombinant DNAs and enzymes, transformants, and methods of making and using the same

The present invention relates to novel DNA, recombinant DNA and DNA-containing enzymes and transformants encoding enzymes that produce non-reducing sugars with trehalose structures as terminal units from reducing starch sugars having a glucose degree of polymerization of 3 or more. The invention also relates to methods and uses for their preparation.

Trehalose is a disaccharide in which glucose 2 molecules are connected to their reducing groups, which are present in extremely small amounts in fungi, algae, and insects. Since trehalose has no reducing residue in the molecule, it does not cause unnecessary browning reaction even when heated in the presence of amino acids, so that sweetening of food can be provided without fear of unnecessary coloring or deterioration. However, trehalose cannot be easily produced in a required amount by a conventional manufacturing method, and is rarely used to sweeten food.

Conventional manufacturing methods are generally divided into two types, one using microbial cells and the other using a multiple enzymatic system that reacts enzymes to sugars. The former method is a method of collecting trehalose from proliferated cells in a culture obtained by growing microorganisms such as bacteria and yeast in a nutrient medium, as disclosed in Japanese Patent Laid-Open No. 154,485 / 75. The latter method prepares maltose as a substrate, as disclosed in Japanese Patent Laid-Open No. 216,695 / 83, and uses a multi-enzyme system using mactose-phosphorylase and trehalose-phosphorylase. To recover the trehalose produced from the reaction system. In the former method, microorganisms can be grown relatively easily, but sequential extraction of trehalose from microorganisms containing only 15 w / w% of trehalose on a dry solid basis (d, s, b,) is performed. There are a number of complex steps. On the other hand, in the latter method, trehalose can be separated relatively easily, but because the enzyme reaction itself is an equilibrium reaction of two different enzymes and the equilibrium point always tilts towards glucose phosphate production, the enzyme is significantly higher in concentration to the substrate. It is theoretically difficult to increase the trehalose yield by

In view of these circumstances, the present inventors have made a thorough search for enzymes that produce trehalose-structured sugars from starch sugars. As a result, Rhizobium sp M-11 and Arthrobacter sp, Q36, etc. It has been found that microorganisms belonging to the species of C are producing new enzymes which produce non-reducing sugars with trehalose structures as terminal units from reducing starch sugars having a glucose degree of polymerization of 3 or more. Before and after this discovery, these non-reducing sugars are hydrolyzed almost quantitatively to another enzyme produced by the same microorganisms as above, producing trehalose and glucose and / or maltooligosaccharides. Combining these enzymes can produce the required amount of trehalose relatively easily, thus completely solving the above-mentioned objectives associated with trehalose. The disadvantage of insufficient production of new enzymes by these microorganisms requires relatively large-scale cultivation to produce non-reducing sugars and / or trehalose with trehalose structures as terminal units on a public scale. .

Recombinant DNA techniques have made significant strides in recent years. Once the gene encoding the enzyme is isolated and the nucleotide sequence is decoded, recombinant DNA having DNA encoding the enzyme is prepared and introduced into the cells of microorganisms or plants and animals. By culturing the enzyme can be easily prepared in the required amount even the enzyme is not stepped on the total amino acid sequence. Under this background, what is urgently needed is to find a gene encoding an enzyme and to identify its nucleotide sequence.

An object of the present invention is to provide a DNA encoding an enzyme for producing a non-reducing sugar having a trehalose structure as a terminal unit from a reducing starch saccharide having a glucose degree of polymerization of 3 or more.

Another object of the present invention is to provide a recombinant DNA containing a DNA and a vector capable of self replication.

It is still another object of the present invention to provide a recombinant enzyme for producing a non-reducing sugar having a trehalose structure as a terminal unit from a reducing starch saccharide having a glucose degree of polymerization of 3 or more by recombinant DNA.

Another object of the present invention is to provide a transformant obtained by introducing a recombinant DNA into a suitable host.

It is a further object of the present invention to provide a method for preparing a recombinant enzyme.

It is another object of the present invention to provide a method for converting reducing starch sugars using a recombinant enzyme.

The first object of the present invention is achieved by DNA encoding enzymes which produce non-reducing sugars with trehalose structures as terminal units from reducing starch sugars having a glucose degree of polymerization of 3 or more.

A second object of the present invention is achieved by self-replicating recombinant DNA containing DNA encoding an enzyme which produces a non-reducing sugar and a vector capable of self-replicating.

A third object of the present invention is achieved by a recombinant enzyme that produces non-reducing sugars with trehalose structure as terminal units from reducing starch sugars having a glucose degree of polymerization of 3 or more.

A fourth object of the present invention is a replication containing a DNA capable of self-replicating DNA encoding an enzyme which produces a non-reducing sugar having a trehalose structure as a terminal unit from a reducing starch saccharide having a glucose degree of polymerization of 3 or more. Possible recombinant DNA is achieved by the transformants obtained by introduction.

A fifth object of the present invention is achieved by a method for producing a recombinant enzyme comprising culturing a transformant capable of producing a recombinant enzyme and extracting the enzyme from the obtained culture.

A sixth object of the present invention is the conversion of reducing starch sugars comprising the step of reacting a recombinant enzyme with reducing starch sugars having a glucose degree of polymerization of 3 or more to produce non-reducing sugars having trehalose structure as terminal units therefrom. Is achieved by the method.

The DNA according to the present invention inserts DNA into a vector capable of appropriate self-replication to generate recombinant DNA that can be replicated, and then introduces a recombinant DNA into a host that can easily reproduce but cannot produce enzymes. To produce a non-reducing glycoenzyme encoded by DNA.

Although recombinant DNA itself does not produce enzymes, production of enzymes encoded by DNA can be achieved by introducing recombinant DNA into a host that cannot produce enzymes, but which can be easily replicated, and culturing transformants to produce enzymes. Induced.

The transformants according to the present invention produce enzymes in culture.

The recombinant enzyme according to the present invention acts on reducing starch saccharides having a degree of glucose polymerization of 3 or more to produce non-reducing sugars whose terminal units are trehalose structures.

The transformant is cultured according to the present invention to obtain the required amount of enzyme relatively easily.

According to the conversion method according to the present invention, a reducing starch saccharide having a glucose degree of polymerization of 3 or more is converted into a non-reducing sugar having a trehalose structure as a terminal unit.

The present invention is based on the discovery of a novel enzyme that produces non-reducing sugars whose terminal units are trehalose structures from reducing starchy sugars having a glucose degree of polymerization of 3 or more. This enzyme was used for rizobium sp. M-11 and Ardrobacter sp. It can be obtained from a culture of microorganisms of Q36 (enzymes from Rizobium sp.M-11 and Ardrobacter Q36 are hereinafter referred to as "enzyme M-11 'and" enzyme Q36 ", respectively).

The present inventors separated the enzyme using conventional purification methods mainly using column chromatography, and then investigated the properties thereof to find the substance, which was a polypeptide having the following physicochemical properties.

(1) action

From non-reducing sugars having a glucose degree of polymerization of 3 or more, non-reducing sugars whose terminal units are trehalose structures are produced.

(2) molecular weight

About 76,000 to 87,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

(3) isoelectric point

3.6 to 4.6 at isoelectric point electrophoresis

(4) optimum temperature

Incubate at pH 7.0 for 60 minutes to show optimum temperature around 35-40 ℃.

(5) optimum pH

Optimum pH of around 6.4-7.2 when incubated at 40 ° C for 60 minutes.

(6) thermal stability

Stable at temperatures below 35 to 40 ° C when incubated at pH 7.0 for 60 minutes.

(7) pH stability

Stable at pH below 5.5 to 11.0 when incubated at 25 ° C for 16 hours.

The experiments performed to elucidate the physicochemical properties above are described in detail below.

Experiment 1: Preparation of Purified Enzyme

Experiment 1-1: Rizobium sp. Preparation of M-11 Derived Enzymes

Liquid containing 2.0 w / v% maltose, 0.5 w / v% peptone, 0.1 w / v% yeast extract, 0.1 w / v% sodium hydrogen phosphate and 0.1 w / v% potassium dihydrogen phosphate in 500 ml Erlenmeyer flask 100 ml each of the medium (pH 7.0) was added thereto, and the flasks were sterilized by autoclaving at 120 ° C. for 20 minutes. The flask was cooled down and the rizobium sp. Seed cultures of M-11 were inoculated into the liquid medium in each flask and then incubated for 24 hours at 27 ° C under rotary shaking conditions. 20 liters of the same fresh liquid medium is placed in a 30 liter jar fermentor and sterilized, and 1 v / v% of the culture broth obtained above is inoculated into a sterilized liquid medium of the ego permuter and pH 6-8 and 30 under aeration and stirring conditions. Incubated for 24 hours at ℃.

Subsequently, about 18 liters of the obtained culture solution was treated in an ultra-high pressure cell disruption apparatus, the cells were crushed, and the obtained suspension was centrifuged to obtain a supernatant. It was left to stand for 1 hour and centrifuged to remove the precipitate. Ammonium sulfate was added to the obtained supernatant, which was allowed to stand at 60 w / v% saturation for 24 hours at 4 ° C. and centrifuged to collect a precipitate, which was dissolved in a very small amount of 10 mm phosphate buffer (pH 7.0). The resulting solution was dialyzed against 10 mM phosphate buffer (pH 7.0) for 24 hours and then centrifuged to remove insoluble material. The supernatant obtained here was passed through a "DEAE-TOYOPEARL" column (product for ion exchange chromatography from Tosoh, Japan) previously equilibrated with 10 mM phosphate buffer (pH 7.0) and then in 10 mM phosphate buffer (pH 7.0). A straight gradient buffer of naturium chloride in the range of 0 M to 0.5 M was passed through the column.The purpose was to collect the fraction containing the enzyme as an eluent and collect it together to collect 50 mM phosphate buffer (pH 7.0) containing 2 M ammonium sulfate. ) Was dialyzed for 10 hours and then centrifuged to remove insoluble material The supernatant obtained was then subjected to 2 M sulfuric acid as a column packed with "BUTYL TOYOPEARL ^* " (for hydrophobic column chromatography from Tosoh, Japan). Straight slope of sodium sulfate in the range of 2 M to 0 M in 50 mM phosphate buffer (pH 7.0) through a column equilibrated with 50 mM phosphate buffer (pH 7.0) containing ammonium Buffer was passed through the column.

Fractions containing the desired enzymes were recovered from the eluate, pooled and pre-equilibrated with 50 mM phosphate buffer (pH 7.0) in a "TOYOPEARL HW-55" column (product for gel filtration column chromatography sold by Tosoh, Japan). After passing through, 50 mM phosphate buffer (PH 7.0) was passed through the column to recover a fraction containing the desired enzyme. The specific activity of this enzyme was about 195 units / mg protein and the yield was about 220 units per liter of culture.

Enzyme activity in the specification is shown by the value measured by the following method. That is, 4 ml of 50 mM phosphate buffer (pH 7.0) containing 1.25 w / v% maltopentaose was added to a test tube, and 1 ml of enzyme solution was added thereto, followed by incubation at 40 ° C. for 60 minutes for enzymatic reaction. Subsequently, the obtained reaction solution was heated at 100 ° C for 10 minutes to stop the enzyme reaction. The obtained reaction solution was diluted 10-fold with distilled water and then the reduction activity was measured by Somogyi-Nelson's method. One unit activity of this enzyme is defined as the amount of enzyme that reduces the reducing power equivalent to 1 u mol of maltopentaose per minute under the same conditions as above.

Experiment 1-2: Purification of Enzyme Q36

As in Experiment 1-1, Arthrobacter sp. The culture broth of Q36 was cultured and the obtained culture was treated to obtain purified enzyme Q36 having an inactivity of about 200 units / mg protein in an yield of about 295 units per liter of culture.

Experiment 2: Physicochemical Properties of Enzyme

Experiment 2-1: Action

Enzyme Q36 obtained in Experiment 1 in 50 mM phosphate buffer (pH 7.0) containing 20 w / v% of glucose, maltose, maltotriose, maltotetraose, maltopentose, maltohexaose or maltoheptaose as substrate. Purified enzyme M-11 was added 2 units / substrate per g of substrate on a dry solids basis (ds b), and the mixture was enzymatically reacted at 40 ° C. for 48 hours. The reaction solution was desalted in a conventional manner and fed to a high performance liquid chromatography (HPLC) column "WB-T-330" (sold by Tosoh, Japan), and the distilled water in this column was flowed at an ambient temperature of 0.5 ml / min. The sugar concentration contained in the reaction solution was separated while the sugar concentration of the eluate was monitored with a differential refractometer "MODEL RI-8012" (manufactured by Japan Chemical Industries, Ltd.). The sugar composition in the reaction solution is shown in Table 1 or Table 2. In the table, the symbols "P1 to P5" are the names given to the sugars produced in the order from the smallest to the largest in the degree of glucose polymerization.

Table 1

TABLE 2

As apparent from the results of Table 1 and Table 2, enzymes M-11 and Q36 are newly sugars from reducing saccharides having a degree of gluose polymerization of 3 or more, such as maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose. On the other hand, glucose polymerization of glucose, maltose and the like does not produce sugars from those less than 3. The newly produced sugars in this enzymatic reaction were P1 to P5, and the total yield of these sugars P2 to P5 was more than 85 w / w% based on the dry solid content.

In order to separate the sugar P1 to P5, three jacketed stainless steel columns (inner diameter 2.0 cm in length 1 m) were charged with the strongly acidic cation exchange resin "XT-1016, Na ⁺ " (sold by Tokyo Organic Chimical Industries, Japan). Connected in series. Reaction liquids containing one sugar among sugars P1 to P5 were separately added to the column at a column internal temperature of 55 ° C., and distilled water at 55 ° C. was added at a flow rate of SV (space velocity) of 0.13. After investigating the sugar composition in the obtained eluate, a fraction containing at least 97 w / w% of one sugar in the sugars P1 to P5 was recovered and pulverized under reduced pressure. By the consumer group-Nelson method, no substantial reducing power was detected in the purified sugars P1 to P5.

To identify sugars P1 to P5, 50 mg of each was weighed, dissolved in 1 ml of 50 mM acetic acid buffer (pH4.5), mixed with 1 unit of glucoamylase, and incubated at 40 ° C for 6 hours. As a result of high performance liquid chromatography analysis on the obtained reaction solution, glucose and trehalose were detected as shown in Tables 3 and 4. When β-amylase was acted on sugars P1 to P5, sugars P1 and P2 were not hydrolyzed by β-amylase, but sugars P3, P4 and P5 were hydrolyzed to yield 1 mole of maltose, 1 mole of sugar P2 and 1 maltose, And P1 and 2 moles of maltose were produced, respectively.

TABLE 3

Note: The molar ratio represented by the symbol "" is a value calculated from the number of moles of glucose per 1 mole of trehalose.

Table 4

Note: The molar ratio represented by the symbol "" is a value calculated from the number of moles of glucose per 1 mole of trehalose.

The results of Table 3 and Table 4 show that sugars P1 to P5 are composed of 1 mol of trehalose and 1-5 mol of glucose. From the fact that glucoamylase specifically hydrolyzes the α-1,4 and α-1,6 bonds of maltooligosaccharides and the β-amylase hydrolyzes the α-1,4 bonds of maltooligosaccharides from the ends to maltose units P1 to P5 have a structure composed of maltooligosaccharides or gluroses having a glucose polymerization degree of 2 to 5, and all of them are considered to have trehalose residues at their ends.

Judging from the above results, the sugars P1-P5 are α-glucosyl trehalose, α-maltosyl trehalose, α-maltotriosyl trehalose, and α-maltotetraosyl trehalose, respectively. It is confirmed that it is loose and α-maltopentaosyl trehalose, and this proves that this enzyme has the activity of producing a non-reducing sugar having a trehalose structure as a terminal unit from reducing sugars having a degree of glucose polymerization of 3 or higher. It is.

Experiment 2-2: molecular weight

U.K. Laemmli Nature, Vol. 227, pp. 680-6685 (1970)], respectively, purifying enzymes M-11 and Q36 in Experiment 1 were subjected to electrophoresis with sodium dodecyl polyacrylamide gel electrophoresis, respectively, at positions corresponding to about 76,000 to 87,000 stone wool. Only one protein band was obtained. The marker proteins used in this experiment were myosin (MW = 200,000 Dalton), β-galactosidase (MW = 116,250 Dalton), phosphorylase B (MW = 97,400 Dalton), serum albumin (MW = 66,200 Dalton) and ovalbumin (MW = 45,000 Dalton).

Experiment 2-3: Isoelectric Point

The isoelectric points by isoelectric point electrophoresis measurements of purified enzymes M-11 and Q36 obtained in Experiment 1 were about 3.6 to 4.6, respectively.

Experiment 2-4: Optimal Temperature

When the purified enzymes M-11 and Q36 obtained in Experiment 1 were incubated in 50 mM phosphate buffer (pH 7.0) for 60 minutes in a conventional manner, the optimum temperature was about 35 to 40 ° C as shown in FIG. .

Experiment 2-5: Optimal pH

The optimum pH when the purified enzymes M-11 and Q36 obtained in Experiment 1 were incubated at 40 ° C. for 60 minutes at different pH of 50 mM acetic acid buffer, phosphate buffer or sodium carbonate-sodium hydrogen carbonate were It was about 6.4-7.2 as shown in FIG.

Experiment 2-6: Thermal Stability

The purified enzymes M-11 and Q36 obtained in Experiment 1 were incubated in 50 mM phosphate buffer (pH 7.0) for 60 minutes to be stable at about 35 to 40 ° C. or lower as shown in FIGS. It was.

Experiment 2-7: pH Stability

When the purified enzymes M-11 and Q36 obtained in Experiment 1 were incubated at 25 ° C. for 16 hours at different pH of 50 mM acetic acid buffer, phosphate buffer or sodium carbonate-sodium hydrogen carbonate buffer, they were tested in a conventional manner. As shown in 8, it was stable at pH about 5.5-11.0 or less.

Experiment 2-8: Arrangement of amino acids with N terminus

The amino acid sequence having the N-terminus of the purified enzyme M-11 obtained in Experiment 1 was analyzed by the gas phase protein sequencer "MODEI. 470A" (sold by Applied Biosytems, USA). It was confirmed that it has an amino acid sequence as shown in 12).

Analysis of the amino acid sequence having the N terminus of the purified enzyme Q36 was performed in the same manner as in the enzyme M-11, and it was confirmed that the amino acid sequence was as shown in the sequence list (SEQ ID NO: 13).

Experiment 2-9: partial amino acid sequence

Appropriate amount of purified enzyme M-11 obtained in Experiment 1-1 was dialyzed against 10 mM Tris-HCI buffer (pH9.0) at 4 ° C for 18 hours, and then mixed with 10 mM Tris-HCI buffer (pH 9.0) for enzyme 1 The concentration was about 1 mg per ml. About 1 ml of the resulting solution was placed in a container, mixed with 10 μg lysyl endopeptidase and incubated at 30 ° C. for 22 hours to partially hydrolyze the enzyme. The hydrolyzate obtained was pre-equilibrated with 0.1 v / v% trifluoro acetate containing an aqueous 16 v / v% acetonitrile solution to a reverse phase high performance liquid chromatography column (Shiseido Co., Ltd., Japan: "CAPCELL-PAK C18"). After the addition, the concentration of acetonitrile was increased from 16 v / v% to 64 v / v%, and 0.1 v / v% trifluoro acetate was fed to the column at a flow rate of 0.9 ml / min. Or fractions with peptide fragments at 40 minutes were separated and recovered (this peptide fractions were referred to as "peptide fragment A" and "peptide fragment B", respectively).

Fractions containing peptide fragments A or B were collected together, dried under reduced pressure, and dissolved in 0.1 v / v% trifluoroacetate containing 50 v / v% acetonitrile. As in Experiment 2-8, peptide fragments A and B were analyzed and found to have amino acid sequences in the sequence list (SEQ ID NO: 14) and amino acid sequences in the sequence list (SEQ ID NO: 15).

As in Enzyme M-11, the enzyme Q36 obtained in Experiment 1-2 was partially hydrolyzed and fed to a column for reverse phase high performance liquid chromatography ("μ BONDAPAK C18" sold by Japan Millipore, Japan), followed by a concentration range of 24 v. 0.1 v / v% trifluoroacetate containing / v% -44 v / v% aqueous acetonitrile was fed to the column at a flow rate of 0.9 mL / min. The fractions containing the peptide fragments eluted at about 22 or about 40 minutes after the start of the supply (these batches are referred to as "peptide fragment C" and "peptide fragment D", respectively), respectively, were collected under reduced pressure and dried under reduced pressure at 50 v. dissolved in 0.1 v / v% trifluoroacetate containing / v% aqueous acetonitrile. Analysis of the peptide fragments C and D was performed in the same manner as above, and it was confirmed that each had an amino acid sequence as shown in the sequence list (SEQ ID NO: 16 and 17).

Enzymes with these physicochemical properties have not been known yet, so it was concluded that this is a novel substance. In the case of rizobium sp, M-11, it is a microorganism isolated from the soil of Okayama City in Japan and deposited on December 24, 1992. (Deposit: Ibaraki, Japan, National Institute of Bioscience and Human -Technology Agency of Industrial Science and Technology and filed under accession number FERM BP-4130. Ardrobacter sp. Q36 is a microorganism isolated from the soil of the city of Soja, Okayama, Japan. It was deposited with the above-mentioned depository institution on June 3, 1993 and stored under accession number FERM BP-4316. Japanese Patent Application No. 349,216 / 93, filed by the present applicant, describes the properties of non-reducing glycoside-producing enzymes and the specific bacteriological properties of these microorganisms.

The present inventors use oligonucleotides chemically synthesized based on the partial amino acid sequence of the enzyme M-11 identified in Experiment 2-9 as probes to produce lyobium sp. A thorough search of the chromosomal DNA of M-11 revealed a 2,316 base pair DNA fragment with base sequence as shown in the following sequence listing (SEQ ID NO: L) starting at the 5 'end. Understanding Decoding of Base Sequences It was confirmed that this enzyme consists of 772 amino acids as shown in the sequence list (SEQID NO: 2).

As in the enzyme M-11, the oligonucleotide chemically synthesized based on the partial amino acid sequence of the enzyme Q36 was used as a probe to search for the chromosomal DNA of the enzyme Q36, and the 5 'terminus as shown in the sequence list (SEQ ID NO: 3). From a DNA fragment having a base sequence consisting of 2,325 base pairs. Decoding this base sequence confirmed that the enzyme Q36 was composed of 775 amino acids and had a partial amino acid sequence with the N terminus as shown in the sequence list (SEQ ID NO: 4). The sequential experimental steps used to identify the amino acid sequence and the nucleotide sequence shown in the sequence list (SEQ ID NO: 1-4) are as follows.

(1) The enzyme was isolated from the culture medium of the donor microorganism and highly purified. The purified enzyme was partially hydrolyzed with protease and the two different peptide fragments obtained were separated to determine their amino acid sequence.

(2) Separately, the chromosomal DNA was isolated from the cells of the donor microorganism, purified, and partially digested with a restriction enzyme to obtain a DNA fragment of about 3,000 to 7,000 base pairs. The DNA fragment was ligated with DNA ligase to a plasmid vector previously cut with a restriction enzyme to obtain recombinant DNA.

(3) The recombinant DNA was introduced into E. coli to obtain a transformant, and the enzyme was isolated from the transformant by a colony hybridization method using oligonucleotides chemically synthesized based on the partial amino acid sequence described above as a probe. A transformant of interest was selected with DNA encoding.

(4) Recombinant DNA was obtained from the transformant and annealed with a primer, followed by the action of a DNA polymerase to extend the primer, and the base sequence of the obtained complementary DNA was determined by dideoxy chain termination. Comparing the amino acid sequence estimated from the determined base sequence with the amino acid sequence shown above confirmed that the base sequence encodes the enzyme.

As described above, enzymes which produce non-reducing sugars with trehalose structures as terminal units from reducing starch sugars having a glucose degree of polymerization of 3 or more are enzymes found by the inventors' long-term research results. This enzyme has physicochemical properties that are distinct from conventional enzymes, and the present invention is prepared by applying recombinant DNA techniques. Recombinant DNA, its preparation method, and its use will be described in detail with reference to Examples.

Recombinant enzyme as used in the present invention refers to a whole enzyme that can be produced by recombinant DNA techniques and can produce non-reducing sugars having trehalose structure as terminal units from reducing starch sugars having a degree of glucose polymerization of 3 or more. In general, the recombinant enzyme according to the present invention has been identified as having an amino acid sequence starting from the N-terminal, such as shown in the sequence list (SEQ ID NO: 2 or 4), and includes homologous to this. A variant with an amino acid sequence homologous to that shown in the sequence list (SEQ ID NO: 2 or 4) may substantially alter one or more bases in the sequence list (SEQ ID NO: 2 or 4) substantially changing the enzyme's inherent action. It can be obtained by substituting with another base. Although the same DNA is used and depends on the components of the host and transformant culture medium into which the DNA is introduced and their culture temperature and pH, they have an amino acid sequence similar to that of the sequence list (SEQ ID NO: 2 or 4) and Intracellular enzymes of the host that have the enzymatic action of the enzyme encoded by the enzyme but lack one or more amino acids located near the N terminus of the amino acid sequence in the sequence list (SE ㅃ ID NO: 2 or 4). It is also possible to produce modified enzymes having one or more newly added amino acids at the N terminus after DNA expression by

Recombinant enzymes can be prepared by culturing transformants with specific DNA. Such a transformant used in the present invention can be prepared by introducing into a host a DNA having a base sequence complementary to, or homologous to, a base sequence starting at the N terminus or gain. Such a base sequence may be prepared by substituting one or more bases using the degeneracy of the genetic code without changing the encoded amino acid sequence. Of course, one or more bases in the base sequence encoding enzymes or variants thereof can be readily substituted with other bases so that the DNA actually expresses enzyme production in the host.

As the DNA used in the present invention, any one having a nucleotide sequence as described above may be used regardless of whether it is naturally derived or artificially synthesized. Examples of natural sources of DNA according to the present invention include Rizobium, Ardrobacter, Brevibacterium, Flavobacterium, Micrococcus and Curtobacterium, Mycobacterium And microorganisms belonging to the genus of Terrabacter and the like, that is, specific examples include lyzobium sp, M-11 (FERM BP-4130), Ardrobacter sp. Q36 (FERM BP-4316), Brevibacterium Hello Volume (ATCC 11822), Flavobacterium Aquatile (IFO 3772), Micrococcus Luteus (IFO: 1064), Micrococcus Roseus (ATCC 186), Curtobacterium Citrium (IFO 15231), Mycobacterium smegmatis (ATCC 19420) and Terrabacter to mesense (IFO 12960), and the like can be obtained genes containing the DNA of the present invention. The microorganisms described above were inoculated in a nutrient medium and incubated for 1 to 3 days under aerobic conditions, and then the obtained cells were recovered from the culture solution and sonicated or treated with cell wall lytic enzymes such as lysozyme or β-glucanase. Extract the gene containing. In this case, proteolytic enzymes such as proteases can be used together with cell wall lysing enzymes, and when the cells are treated with an ultrasonic crusher, they are treated in the presence of a surfactant such as sodium dodecyl sulfate (SDS), or frozen or thawed. You may process it. The desired DNA is produced by treating the obtained product by a method commonly used in the art, such as phenol extraction, alcohol precipitation, winsim separation, protease treatment, and / or ribonuclease treatment. Artificially synthesizing the DNA of the present invention chemically synthesized using the base sequence in the sequence list (SEQ ID NO: 1 or 3), or the amino acid sequence in the sequence list (SEQ ID NO: 2 or 4) Inserting the DNA into a vector capable of self-replicating to obtain recombinant DNA, introducing the recombinant DNA into a suitable host to obtain a transformant, and then cultivating the transformant to isolate the proliferated cells from the culture obtained. From these cells, a plasmid containing DNA can be obtained in the form of a plasmid.

Such recombinant DNA is generally introduced into the host in the form of recombinant DNA. In general, recombinant DNA contains a DNA capable of self-replicating with the above-mentioned DNA, which can be produced relatively easily by recombinant DNA techniques when the DNA raw material is secured. Examples of such vectors include plasmid vectors such as pBR322, pUC18, Bluescript II SK (+), pUB110, pTZ4, pC194, pHV14, TRp7, TEp7, pBS7, and λ gt λ C, λ gt · λ B, There are phage vectors such as p 11, Φ 1, and Φ 105. Among these plasmid vectors and phage vectors, pBR322, pUC18, Blueseript II SK (+), λ gt · λ C and λ gt λ B can be used satisfactorily when the DNA of the present invention needs to be expressed in Escherichia coli, whereas pUB110, pTZ4, pC194, p 11, Φ 1 and Φ 105 are used to express DNA in Bacillus microorganisms. It can be used satisfactorily. Plasmid vectors pHV14, TRp7, TEp7 and pBS7 are advantageously used when growing recombinant DNA in two or more hosts.

In the present invention, the method of inserting the DNA of the present invention into such a vector may be a conventional method commonly used in the art. The gene containing the DNA of the present invention and the self-replicating vector are first digested with restriction enzymes and / or ultrasonic disrupters, and then ligation of the obtained DNA fragments and vector fragments is performed. Degradation of DNA and vector involves nucleotides, in particular type II restriction enzymes, more specifically restriction enzymes that specifically act on Sau 3AI, Eco RI, Hind III, Bam HI, SaI I, Xba I, Sac I, Pst I, etc. DNA fragments and vector fragments can be easily ligated. To ligate DNA fragments into vector fragments, they are annealed as needed and then treated with DNA ligase in vivo or in vitro. The recombinant DNA thus obtained can be replicated without any limitation by culturing the transformant obtained by introducing into an appropriate host.

The recombinant DNA obtained here can be introduced into suitable host microorganisms including Escherichia coli, Bacillus genus, Actinomyces genus and yeast. In case of using E. coli as a host, DNA can be introduced by culturing the host in the presence of recombinant DNA and calcium ions, and when using Bacillus microorganism as a host, the competent cell method and colony A hybrid hybridization method can be used. Cloning the required transformants by colony hybridization or culturing various transformants in a nutrient medium containing reducing starch sugars with a glucose degree of polymerization of 3 or more, and then trehalo as terminal units from the reducing starch sugars. It can be cloned by selecting a target transformant that produces a non-reducing starchy saccharide having an os structure.

The transformant obtained in this way produces the desired enzyme extracellularly when cultured in a nutrient medium. In general, a liquid medium supplemented with a carbon source, a nitrogen source and minerals, and a liquid medium supplemented with a small amount of amino acids and vitamins, if necessary, may be used in the present invention. Examples of carbon sources include sugars such as starch, starch hydrolyzate, glucose, fructose and sucrose, and examples of nitrogen sources include ammonia, ammonium salt, urea, nitrate, peptone, yeast extract, skim soybean, corn leachate, meat extract, etc. Nitrogen-containing inorganic and organic materials. A culture solution containing the target enzyme can be prepared by inoculating a transformant in a nutrient medium and incubating at 25 to 65 ° C. and pH 2 to 8 for about 1 to 6 days under aerobic conditions with aeration and agitation. The culture solution can be used as an enzyme as it is, and usually, the enzyme is separated from cells and debris by filtration and / or centrifugation by ultrasonic crusher and / or cell wall fusion enzyme, and the enzyme is purified and used. Enzyme purification methods generally include conventional methods. Remove cells and cell debris from the culture and use one of the following methods: centrifugation, salting out, dialysis, fractional precipitation, gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel electrophoresis, and isoelectric electrophoresis. Process as above.

As described above, the recombinant enzyme according to the present invention is characterized by producing non-reducing sugars having trehalose structure as terminal units from reducing starch sugars having a glucose degree of polymerization of 3 or more. The resulting non-reducing sugars have a mild high quality sweetness, moderate viscosity and moisturizing properties, and since they do not have reducing moieties in their molecules, they can sweeten foods without fear of coloring or deterioration. This feature can be used to convert various starch sugars that have become obsolete for reducing reasons to sugars that are satisfactory, useful, and substantially free or extremely reduced in their handling.

The switching method will be described in detail below. Reductive starch hydrolysates obtained by partial hydrolysis of starch, amylopectin and amylose with acids and / or amylases can typically be used as substrates of the recombinant enzymes of the invention. Such starch hydrolyzate can be prepared by conventional methods, for example, at least one of maltooligosaccharides having a glucose degree of polymerization of 3 or more, that is, maltotriose, maltotetraose, maltopentaose, mactohexaose and maltoheptaose. This includes. Α-amylase, maltotetraose production, as described in "Handbook of Amylases and Related Enzymes", lst edition, edited by Thc Amylase Research Society of Japan, published by Pergamon Press plc, Oxford, England (1988). Amylase, maltopentaose producing amylase and maltohexaose producing amylase are particularly useful in the preparation of reducing starch sugars used in the present invention, even when using one of these amylases, they contain large amounts of reducing starch sugars having a glucose degree of polymerization of 3 or more. One starch sugar mixture is obtained in a fairly high yield. If necessary, starch debranching enzymes such as pullulanase and isoamylase and amylase can increase the yield of reducing starch saccharides used as substrates of the recombinant enzyme of the present invention.

In the conversion method according to the present invention, a recombinant starch sugar of the present invention is co-existed in an aqueous solution containing at least one of the above-mentioned reducing starch sugars as a substrate, and the solution is subjected to enzymatic reaction at a predetermined temperature and pH. Produce it in the required amount. Although the enzymatic reaction proceeds at a substrate concentration of less than 0.1 w / v%, using a high concentration of 2 w / v%, preferably 5-50 w / v%, the conversion method according to the present invention can be applied to industrial production scale. It can be applied satisfactorily. The temperature and pH used for this enzymatic reaction are set within a range in which the recombinant enzyme is effectively acted on the substrate without inactivating the recombinant enzyme, that is, a temperature of about 55 ° C., preferably about 40 to 55 ° C. and a pH of 5 to 10 Preferably, it is set in the range of pH 6-8. The amount and reaction time of the recombinant enzyme of the present invention are selected differently depending on the enzyme reaction conditions. The enzymatic reaction significantly reduces the reducing power of reducing starch sugars with a glucose degree of polymerization of 3 or more, which, in the case of maltopentaose, is reduced by about 7% relative to the initial level.

The reaction mixture obtained in the conversion reaction of the present invention can be used as it is, usually after purification. That is, the insoluble material is removed from the reaction mixture by filtration and centrifugation, and the obtained solution is decolorized by deactivation with activated charcoal, and then purified by ion exchanger and concentrated to obtain a syrupy product. Depending on the application, this syrup-like product is dried under reduced pressure and spray dried to obtain a solid product. To prepare products of non-reducing sugars, the syrup-like products shown above may be subjected to chromatography using ion exchangers, separation of sugars by activated carbon and silica gel, fractional precipitation using alcohol and / or acetone, membrane filtration and yeast. Treatment is carried out by one or more of the methods such as fermentation by, and removal and decomposition of reducing sugars by alkali. For example, as a method of treating a large amount of reaction mixture, there is a fixed phase method or pseudo mobile phase method-ion exchange column chromatography (see Japanese Patent Laid-Open Nos. 23,799 / 83 and 72,598 / 83). High-reducing sugar-containing products can be produced on industrial scale at significantly higher yields.

The reducing sugars thus obtained have a wide range of uses in various products which are easily damaged by the reducing properties of sugar sweeteners. For example, it can be satisfactorily used as a sweetener, a taste improver, a quality improver, a stabilizer, a filler, an excipient and an adjuvant in food, cosmetics and pharmaceuticals. Non-reducing sugars produce trehalose almost quantitatively when subjected to the enzymatic action of trehalose releasing enzyme disclosed in Japanese Patent Application No. 340,343 / 92. Can be used.

The present invention is described in more detail in the following examples, where recombinant DNA techniques or other techniques themselves are used in the art, such as in J, Sumbruck et al. in "Molecular Cloning A Laboratory Manual", 2nd edition, published by Cold Spring Harbor Laboratory Press, USA (1989).

Example 1 Preparation of DNA-Containing Recombinant DNA and Convertors Derived from Enzyme M-11

Example 1-1 Preparation of Chromosome DNA

Rizobium sp. Seed cultures of M-11 were inoculated in bacteriotrophic broth medium (pH 7.0) and incubated in a rotary shaker at 27 ° C for 24 hours. The cells were removed by centrifugation from the culture, suspended in TES buffer (pH 8.0), mixed with 0.05 w / v% lysozyme, and incubated at 37 ° C. for 30 minutes. The obtained product was frozen at −80 ° C. for 1 hour, mixed by addition of TSS buffer (pH 9.0), heated to 60 ° C., mixed with a mixture of TES buffer and phenol, quenched with ice, and centrifuged to precipitate. One crude chromosomal DNA was recovered. Two times the volume of cold ethanol was added to the supernatant, and the precipitated crude chromosomal DNA was recovered, suspended in SSC buffer (pH 7.1), mixed with 7.5 μg of ribonuclease and 125 μg of protease, and incubated at 37 ° C. for 1 hour. . Subsequently, a mixture of chloroform and isoamyl alcohol was added to the reaction solution to extract the target chromosomal DNA, mixed with cold ethanol, and the chromosome DNA-containing precipitate was collected. Purified chromosomal DNA obtained here was dissolved in SSC buffer (pHl 7.1) to a concentration of about 1 mg / ml and the solution was frozen at -80 ° C.

Example 1-2 Preparation of Recombinant DNA pBMT7 and Transformant BMT7

About 1 ml of the purified chromosomal DNA prepared in Example 1-1 was added to a container, mixed with about 35 units of Sau 3AI, a restriction enzyme, and mixed for about 20 minutes at 37 ° C. for partial digestion of chromosomal DNA. By centrifugation, DNA fragments of about 3,000 to 7,000 base pairs were recovered. 1 μg of the plasmid vector Bluewooscript II SK (+) was prepared, and the restriction enzyme Bam HI was used to completely digest the plasmid vector, followed by mixing with 10 μg of DNA fragment and 2 units of T4 DNA ligase and overnight at 4 ° C. The DNA fragment was ligated to the vector fragment by standing. 30 μl of “Epicurian Coli ^* XLI-Blue”, (competitor cell sold by Toyobo, Japan) was added to the obtained recombinant DNA, and the mixture was left to stand for 30 minutes under ice cooling conditions, heated to 42 ° C., and then mixed with SOC broth. The recombinant DNA was introduced into E. coli by incubation at 37 ° C. for 1 hour.

The resulting transformants were inoculated in agar plates (pH 7.0) containing 50 μg / ml of 5-bromo-4-chloro-3-indolyl-β-galactoside, incubated at 37 ° C. for 18 hours, and then agar plates. Nylon film was placed on top of this to fix about 4,400 colonies produced on the agar plate. Based on the amino acid sequence Pro-Glu-Trp-Glu-Lys at positions 17-21 of the amino acid sequence of peptide fragment A identified in Experiment 2-9, the probe 1 of sequence 1 (SEQ ID NO: 5) The base sequence was chemically synthesized, labeled with ³² P, hybridized with colonies of transformants immobilized on nylon films, and nine transformants with intense hybridization were selected.

From these nine transformants, the recombinant DNA of interest was selected as the conventional method, and described in E.M. Southern in Journal of Molecular Biology, Vol. 98, pp. 503 to 517 (1975), based on the amino acid sequence Thr-Glu-Phe-Trp-Asp at positions 16-20 of the amino acid sequence of peptide fragment B identified in Experiment 2-9 Was hybridized with Probe 2 having the base sequence in the chemically synthesized sequence list (SEQ ID NO: 6), and then recombinant DNA strongly hybridized with Prove 2 was selected. The recombinant DNA and transformant thus selected were named pBMT7 and BMT7, respectively.

The transformant BMT7 was inoculated in L-juice (pH 7.0) containing 100 μg / ml of ampicillin, incubated in a rotary shaker at 37 ° C. for 24 hours, and the cells were collected from the culture medium by centrifugation, followed by alkaline method. Treatment was performed to extract recombinant DNA extracellularly. Purification and analysis of the obtained product in a conventional manner confirmed that the recombinant DNA pBMT7 was composed of about 9,300 base pairs and had a structure represented by a restriction map as shown in FIG. 9. In addition, as shown in FIG. 9, it was confirmed that the DNA consisting of 2,316 base pairs encoding the enzyme M-11 was located downstream of the degradation site by the restriction enzyme Pst I.

Example 1-3 Preparation of Enzyme by Transformant

Adjust liquid phase to pH 7.0 with 2.0 w / v% maltose, 0.5 w / v% peptone, 0.1 w / v% yeast extract, 0.1 w / v% sodium dihydrogen phosphate and 0.1 w / v% potassium dihydrogen phosphate 50 μg / ml of ampicillin was added thereto, mixed, autoclaved at 120 ° C. for 20 minutes, cooled, inoculated with the seed culture medium of the transformant BMT7 obtained in Example 1-2, and then shaken at 37 ° C. for 24 hours. The transformants were cultured in the phase. The obtained culture was treated with an ultrasonic crusher to disrupt cells, and the obtained suspension was centrifuged to remove insoluble matters. As a result of measuring the enzyme activity with respect to the obtained supernatant, about 3,000 units of enzyme were obtained from 1 liter of this culture solution.

As a control, seed culture medium of E. coli XLI-blue cow or risobium sp, M-11 was inoculated into fresh fresh liquid medium containing no ampicillin, and in the case of culture medium of risobium sp, M-11, this was 30 ° C. At the incubation temperature of the culture was carried out as above. As a result of analyzing the activity of the obtained product, lyzobium sp. About 1,500 units of enzyme were obtained from 1 L of culture medium of M-11, and the yield was significantly lower than that of the transformant BMT7. E. coli XLI-blue cows used as hosts did not produce enzymes.

Subsequently, the enzyme produced by the transformant BMT7 was purified in the same manner as in Experiment 1-1, and the properties thereof were examined. It was confirmed to have substantially the same physical and chemical properties as in Experiment 2. In addition, it can be seen from this result that the enzyme of the present invention can be produced by recombinant DNA technique, and thus the yield is considerably increased.

Example 2 Preparation of Complementary DNA Derived from Enzyme M-11 and Determination of its Base Sequence and Amino Acid Sequence

2 μg of the recombinant DNA pBMT7 prepared by the method of Example 1-2 was mixed with a 2 M aqueous sodium hydroxide solution, denatured, mixed with an appropriate amount of cold ethanol, and the resulting template DNA-containing precipitate was collected and dried under reduced pressure. . Chemically synthesized primer 1 with base sequence of sequence list (SEQ ID NO: 7) in this template DNA 1 40 pm Tris-HCI buffer containing 50 pmole / ml and 20 mM magnesium chloride and 50 mM sodium chloride, pH 7.5 Μl was added and incubated for 2 min at 65 ° C., followed by annealing, and the mixture was then dATP, dGTP and dTTP in 7.5 μM, [α- ^32- P] dCTP, respectively, 0.5 μl (2 mCi / ml), 0.1 2 μl of an aqueous solution containing 1 μl M dithiothreitol and 2 μl 1.5 units / ml T7 DNA polymerase followed by incubation at 25 ° C. for 5 minutes to extend primer 1 from the 5 ′ end to the 3 ′ end. Complementary chain DNA was generated.

Dilute the reaction product containing the complementary chain DNA into quarter portions and add 2.5 μl of 50 mM aqueous sodium chloride solution containing 80 μM dNTP and 8 μM ddATP, ddCTP, ddGTP, ddGTP or ddTTP to each of them. Incubate at 37 ° C. for 5 minutes and stop the reaction by adding 4 μl of an aqueous 95 v / v% formamide solution containing 20 mM EDTA, 0.05 w / v% bromophenol blue cow and 0.05 w / v% xylene cyanols. I was. The reaction mixture was placed in a vessel and heated in a boiling water-bath for 3 minutes and then added to a gel containing 6 w / v% polyacrylamide, followed by activation of the gel at a constant voltage of about 2000 volts, followed by electrophoresis to DNA fragments. The gels were separated and the gels were fixed in the usual manner, dried and treated by autoradiography.

Analysis of DNA fragments isolated from the radiographs revealed that the complementary chain DNA had a base sequence consisting of 2.936 base pairs as shown in the sequence list (SEQ ID NO: 10). The amino acid sequence deduced from this base sequence is shown in the sequence list (SEQ ID NO: 10), which is the partial amino acid sequence of enzyme M-11 in the sequence list (SEQ ID NO: 12, 14, or 15). As compared with the amino acid sequence having N and N termini, the N terminal amino acid sequence of the sequence list (SEQ ID NO: 12) corresponds to the amino acid sequence at positions 1-20 of the sequence list (SEQ ID NO: 10). The partial amino acid sequence of (SEQ ID NO: 14 or 15) was confirmed to correspond to the amino acid sequence at positions 606 to 626 or 486 to 506 of the sequence list (SEQ ID NO: 10). From these results, the enzymes produced in risobium sp, M-11 have the amino acid sequence of the sequence list (SEQ ID NO: 2), and the enzymes derived from the microorganism have the base sequence in the sequence list (SEQ ID NO: 1). It can be seen that it is encoded by the DNA having.

Example 3: Artrobacter sp. Preparation of recombinant DNA and transformants containing DNA from Q36

Example 3-1 Preparation of Chromosome DNA

As in Example 1-1, Arthrobacter sp. Chromosomal DNA was isolated and purified from Q36, dissolved in SSC buffer (pH 7.1) to a concentration of about 1 mg / ml and the solution was frozen at -80 ° C.

Example 3-2 Preparation of Recombinant DNA pBQT13 and Transformant BQT13

The purified chromosomal DNA obtained in Example 3-1 was partially digested in the same manner as in Example 1-2, and the DNA fragment consisting of about 3,000 to 6,000 base pairs was recovered by sucrose density gradient ultracentrifugation. This DNA fragment was ligated to the cell lysate of Bluewooscript II SK (+) treated with Bam HI as in Example 1-2, and the resulting recombinant DNA was introduced into Escherichia coli XLI-Bluewool. The transformants obtained here were cultured in agar plates containing 5-bromo-4-chloro-3-indolyl-β-D-galactosidate as in Example 1-2, and the resulting about 4,500 colonies were nylon While fixed on the film, probe 3 with the nucleotide sequence in the sequence list (SEQ ID NO: 8) is located at positions 11-16 of the amino acid sequence of peptide fragment D in the sequence list (SEQ ID NO: 17). and Phe-Asp-Val-Asp- Trp-Asp , based on the amino acid sequence expressed by a chemically synthesized and then after hybridizes to ³² P cover the transformant colonies which had been fixed on the nylon film by the probe 3 Eight transformants that were strongly hybridized were selected.

As in Example 1-2, the target recombinant DNA was selected from eight transformants, and the amino acid sequence at position 16-20 in the sequence list (SEQ ID NO: 16), that is, Thr-Glu-Phe-Trp Based on -Asp, hybridized with Probe 4 having the nucleotide sequence shown in the chemically synthesized sequence list (SEQ ID NO: 9), and then recombinant DNA that was strongly hybridized with Probe 4 was selected. The recombinant DNA and transformant thus selected were named pBQT13 and BQT13, respectively.

After incubating the transformant BQT13 in L broth containing ampicillin and culturing in the same manner as in Example 3-2, the proliferated cells were recovered, and the recombinant DNA was extracted, purified and analyzed. It was confirmed that it is composed of about 7,200 base pairs and has a structure represented by the limit shown in FIG. As shown in FIG. 3, it can be seen that the DNA consisting of 2,325 base pairs and encoding the DNA of the enzyme Q36 is located downstream of the cleavage site of Xmn I.

Example 3-3 Preparation of Enzyme by Transformant BQT13

Adjust liquid medium to pH 7.0 with 2.0 w / v% maltose, 0.5 w / v% peptone, 0.1 w / v% yeast extract, 0.1 w / v% sodium hydrogen phosphate and 0.1 w / v% potassium dihydrogen phosphate 50 μg / ml of ampicillin was added thereto, mixed, autoclaved at 120 ° C. for 20 minutes, cooled, inoculated with the seed culture solution of the transformant BQT13 obtained in Example 3-2, and then shaken at 37 ° C. for 24 hours. The transformants were cultured in the phase. The obtained culture was treated with an ultrasonic crusher to disrupt cells, and the obtained suspension was centrifuged to remove insoluble matters. As a result of measuring enzyme activity on the obtained supernatant, about 2,450 units of enzyme were obtained from 1 liter of this culture solution.

As a control, the seed culture solution of Escherichia coli XLI-blue cow or Ardrobacter sp, Q36 was inoculated in fresh fresh liquid medium containing no ampicillin, and treated by incubating as above at a culture temperature of 30 ° C. As a result of analyzing the activity of the obtained product, about 1,200 units of enzyme were obtained from 1 L of the culture solution of Ardrobacter sp, Q36, and the yield was significantly lower than that of the transformant BQT13. E. coli XLI-blue cows used as hosts did not produce enzymes.

Subsequently, the enzyme produced by the transformant BMT7 was purified in the same manner as in Experiment 1-1, and the properties thereof were examined. As a result, it showed an molecular weight of about 76,000 to 87,000 Daltons on SDS-PAGE and an isoelectric point of about 3.6 to 4.6 on isoelectric electrophoresis. It was confirmed to have substantially the same physical and chemical properties as in Experiment 2.

In addition, it can be seen from this result that the enzyme of the present invention can be produced by recombinant DNA technique, and thus the yield is considerably increased.

Example 4 Artrobacter sp. Preparation of complementary chain DNA derived from Q36 and determination of its nucleotide sequence and amino acid sequence

The recombinant DNA pBQT13 obtained in Example 3-2 was treated in the same manner as in Example 2 to generate running DNA, and then annealed with Primer 1, followed by T7 DNA polymiraase, to act from 5 'to 3' ends. Complementary chain DNA was obtained by extending primer 1. As in Example 2, complementary chain DNA was treated with dideoxy chain termination to analyze DNA fragments separated on radiographs. This result shows that the complementary chain DNA has a nucleotide sequence consisting of 3,073 base pairs, and the amino acid sequence deduced from the nucleotide sequence is as shown in the sequence list (SEQ ID NO: 11). This amino acid sequence is compared with the partial amino acid sequence of the sequence list (SEQ ID NO: 13, 16 or 17) and the N terminal amino acid sequence. As a result, the N terminal amino acid sequence of the sequence list (SEQ ID NO: 13) is Corresponding to positions 1-20 of ID NO: 11, the partial amino acid sequence of the sequence list (SEQ ID NO: 16 and 17) is 110-129 position or 606-625 of the sequence list (SEQ ID NO: 11). It was confirmed that it corresponds to the amino acid sequence in position. The results also show that the enzyme Q36 has an amino acid sequence of the sequence list (SEQ ID NO: 4), which indicates that the base sequence in the sequence list (SEQ ID NO: 3) is encoded by the DNA having it.

Example 5 Preparation of Recombinant Enzymes

Liquid medium with 2.0 w / v% maltose, 0.5 w / v% peptone, 0.1 w / v% yeast extract, 0.1 w / v% sodium hydrogen phosphate and 0.1 w / v% potassium dihydrogen phosphate in a 500 ml Erlenmeyer flask (pH 7.0) 100 ml each was added, and 50 µg / ml of ampicillin was added to each flask, followed by autoclave treatment at 120 ° C for 20 minutes. Each flask was then cooled and inoculated with the transformant BMT7 obtained in Example 1-2, followed by incubating the transformant on a rotary shaker at 27 ° C. for 24 hours. Separately from this, 18 liters of the same freshly prepared liquid medium was added to an Erlenmeyer flask, 50 μg / ml of ampicillin was added, sterilized at 120 ° C. for 20 minutes, cooled, inoculated with 1 v / v% of the culture medium obtained above, and then aerated. Incubated for 24 hours at 37 ℃ under stirring. The obtained culture was treated with an ultrasonic crusher to disrupt cells, and the obtained suspension was centrifuged to remove insoluble matters. As a result of measuring the enzyme activity with respect to the obtained supernatant, about 3,000 units of enzyme were obtained from 1 liter of this culture solution. The supernatant was purified by the method of Experiment 1-1 to obtain about 50 ml of an aqueous solution containing about 135 units / ml of a recombinant enzyme having an activity of about 200 units / mg of protein.

Example 6: Preparation of Recombinant Enzymes

The recombinant transformant BQT13 prepared by the method of Example 3-2 was cultured in the same manner as in Example 5, and the obtained culture was treated with an ultrasonic crusher to disrupt cells. The obtained suspension was centrifuged to remove insoluble matters and the enzyme activity was measured on the obtained supernatant, resulting in about 2,450 units of enzyme per liter of culture. The supernatant was purified by the method of Experiment 1-1 to obtain about 45 ml of an aqueous solution containing about 120 units / ml of a recombinant enzyme having an activity of about 200 units / mg of protein.

Example 7 Conversion of Starch Hydrolysates by Recombinant Enzymes

Potato starch was suspended in water to make a 6 w / w% suspension which was autoclaved at 120 ° C. for 10 minutes to gelatinize the starch. The gelatinized starch was rapidly cooled to 50 ° C. and adjusted to a pH of about 4.5 to mix the isoamylase product (sold by Hayashibara Biochemical Laboratories, Japan) with 2,500 units per 1 g of starch on a dry solids basis, followed by enzymes at 50 ° C. for 20 hours. Reacted. The reaction mixture was adjusted to pH 6.0, autoclaved at 120 ° C. for 10 minutes to inactivate the remaining enzymes, and then rapidly cooled to 45 ° C., the α-amylase product "TERMAMYL 60L" (Sold by Novo Nordisk Bioindustri A / S, Denmark). Was mixed with 150 units per 1 g of starch on a dry solids basis and then subjected to enzymatic reaction at 45 ° C. for 24 hours to contain a reducing starch sugar such as maltotriose, maltotetraose and maltopentaose having a glucose degree of polymerization of 3 or more. Got. The reaction mixture was autoclaved at 120 ° C. for 20 minutes to inactivate the remaining enzymes and was quenched to 45 ° C., whereby the recombinant enzyme obtained in Example 5 was added to 1 unit per 1 g of starch on a dry solids basis and mixed at 96 ° C. for 96 hours. Reacted. The resulting reaction mixture was heated at 96 ° C. for 10 minutes to inactivate the remaining enzyme, cool, and filtered, and then the filtrate was decolorized with activated carbon in a conventional manner. After desalting, purification with an ion exchanger, and concentration were carried out to obtain about 70 w / w% syrup on the basis of dry solids in a yield of about 91% (dry solids).

From the analytical results of the syrup conducted in Experiment 2-1, DE (Dextrose Equivalent: dextrose eqivalent) of this product was about 18.7 and 8.4 w / w% α-glucosyl trehalose, 5.6 w on a dry solids basis. / w% α-maltosyl trehalose, 37.9 w / w% α-maltotriosyl trehalose as the main component, most of the reducing sugars listed above are converted to their respective non-reducing sugars It was confirmed. It has a mild, moderate sweetness, moderate viscosity and moisturizing properties. It can be used satisfactorily as a sweetener, taste improver, quality improver, stabilizer, filler, excipient and supplement in foods, cosmetics and pharmaceuticals. This product contains a relatively large amount of non-reducing sugars and can be used as an intermediate in the preparation of trehalose.

Example 8 Conversion of Starch Hydrolysates by Recombinant Enzymes

Potato starch was suspended in water to a concentration of 33 w / w% on a dry solids basis and mixed with 0.1 w / w% calcium carbonate on a dry solids basis. To the obtained suspension, 0.2 w / w% of α-amylase product "TERMAMYL 60L" (Sales of Novo Nordisk Bioindustri A / S, Denmark) was added per 1 g of starch on a dry solid basis, followed by enzymatic reaction at 95 ° C for 15 minutes. I was. The reaction mixture was autoclaved at 120 ° C. for 10 minutes to inactivate residual enzymes, followed by quenching and drying of maltotetraose producing enzymes derived from Pseudomonas stutzeri disclosed in JP-A-240,784 / 88. 5 units per 1 g of starch were added and mixed on a solids basis, followed by enzymatic reaction at 55 ° C. for 6 hours. Subsequently, the obtained reaction mixture was mixed with 30 units of α-amylase standard “α-amylase 2A” (sold by Ueda Chemical Co., Ltd., Japan) per 1 g of starch on a dry solid basis and subjected to enzymatic reaction at 65 ° C. for 4 hours to determine the degree of glucose polymerization. About 50 w / w% (dry solids) of reducing starch sugars such as maltotriose, maltotetraose, and maltopentose, which are three or more, were produced. The resulting mixture was autoclaved at 120 ° C. for 10 minutes to inactivate the remaining enzymes, quench to 45 ° C. and adjust to pH 4.5 to add 2 units per 1 g of starch based on dry solids. After mixing, the reaction was carried out at 45 ° C. for 64 hours. The resulting mixture was heated at 95 ° C. for 10 minutes to inactivate the remaining enzymes, cool, filter, decolorized with activated carbon, desalted, purified by ion exchanger, and concentrated to a concentration of about 70 w / w% on a dry solids basis. The syrup-like product of was obtained in a yield of about 90% based on dry solids relative to starch as a raw material.

As a result of analyzing the syrup phase product by the method of Experiment 2-1, its DE was 10.5 and based on dry solids, 3.8 w / w% α-glucosyl trehalose, 43.8 w / w% α-maltosyl trehalo Os and 1.2 w / w% α-maltotriosyl trehalose were the main components, and it was confirmed that most of the reducing starch sugars contained therein were converted to their respective non-reducing sugars. This product has mild moderate sweetness, moderate viscosity and moisturizing properties, so it can be used satisfactorily as a sweetener, taste improver, quality improver, stabilizer, filler, excipient and supplement in food, cosmetics and medicines. It contains a relatively high content of non-reducing sugars and can be used as an intermediate in the preparation of trehalose.

Example 9 Conversion of Maltopentaose by Recombinant Enzyme

High purity maltopentaose (manufactured by Hayashibara Biochemical Laboratories, Japan) was dissolved in water to a concentration of 20 w / w% (based on dry solids), and the solution was adjusted to pH 6.5 to dry the recombinant enzyme prepared in Example 5. 1 unit per 1 g of maltopentaose was added and mixed on a solids basis, followed by enzymatic reaction at 45 ° C. for 48 hours. The reaction mixture was heated at 95 ° C. for 10 minutes to inactivate the remaining enzyme, cool, filter, and concentrated and analyzed by the method in Experiment 2-1. As a result, about 92 w / w% of maltopentaose as a raw material (based on dry solids) It was confirmed that was converted to α-maltotriosyl trehalose.

Four jacketed stainless steel columns (5.4 cm in diameter and 5 m in length) are uniformly filled with strong acid cation exchange resins ("XT-1016 (Na type)" sold by Tokyo Organic Chemical Industries, Japan) and connected in series. The total length of the column was 20 m. The reaction mixture obtained above was fed into the column at 55 ° C. in the column at a flow rate of about 5 v / v% to the resin, and then the column was supplied with SV (space velocity) of 0.13 to 55 ° C. to elute the sugar component. I was. According to the analysis of the sugar composition in the eluate, a high fraction of non-reducing sugars was collected, collected, concentrated, and dried under reduced pressure to obtain a solid powder in a yield of about 55% based on dry solids.

From the results of analyzing the solid product by the method of Experiment 2-1, it was confirmed that its DE was less than about 0.2 and contained 99.0 w / w% α-maltotriosyl trehalose on a dry solids basis. This product has relatively low hygroscopicity, low reducibility, and a little sweetness, so it can be used satisfactorily as a sweetener, taste improver, quality improver, stabilizer, filler, excipient, and auxiliary tax in food, cosmetics and pharmaceuticals. In addition, this product has a relatively high content of non-reducing sugars and can be used as an intermediate in the preparation of trehalose.

Example 10 Conversion of Starch Hydrolysates by Recombinant Enzymes

"PINE-DEX # 4" (starch hydrolyzate from Matsutani Chemical Ind. Of Japan)

Was dissolved in water to a concentration of 40 w / w% on the basis of dry solids, this solution was heated to 45 ℃ adjusted to pH 6.5 and the recombinant enzyme prepared in Example 5 1 g of starch hydrolyzate on the basis of dry solids 1 unit of sugar was added and mixed, followed by enzymatic reaction at 45 ° C. for 96 hours to prepare a reaction mixture containing non-reducing sugar having a trehalose structure as a terminal unit. The reaction mixture was then heated at 100 ° C. for 10 minutes to inactivate and concentrate the remaining enzyme, yielding a solution of up to 20 w / w% on a dry solids basis, then cooling to 55 ° C. and adjusting to pH 4.5 to provide a glucoamylase product, “GLUCOZYME”. "(Sales of Nagase Biochemicals, Japan) was added to 1 g of sugar on a dry solid basis and mixed for 10 hours, followed by enzymatic reaction for 40 hours. The reaction mixture was heated at 100 ° C for 10 minutes to inactivate and cool the remaining enzyme Decolorized with activated carbon in a conventional manner, desalted, purified with ion exchanger, and concentrated to give about 60 w / w% syrup-like product containing about 29.7 w / w% trehalose on a dry solids basis.

Except for using "CG 6000 (Na ⁺ type)", the syrup-like product was fractionated in the same manner as in Example 9, and a fraction containing about 90 w / w% trehalose based on dry solids was collected and concentrated to a concentration. Obtain a 75 w / w% solution, put it in a crystallizer, add about 2 w / w% trehalose hydrate on the basis of dry solids as a seed to saccharides, mix, and crystallize with gentle stirring to crystallize with a crystallinity of about 45%. A kit (massecuite) was obtained. A belt installed at the bottom of the spray tower by spraying this masquite downwards through the nozzle installed at the top of the spray tower at a pressure of about 150 kg / cm ² while blowing hot air at about 85 ° C. from the top of the spray tower. Crystal powder was accumulated on the conveyor and then slowly discharged from the drying tower. Thereafter, the powder was introduced into the scouring tower and aged for 10 hours while blowing the hot air of about 40 ° C to the contents to complete crystallization and drying.

This product is practically non-hygroscopic, mild and has high quality sweetness, so it can be used satisfactorily as a sweetener, taste improver, quality improver, stabilizer, filler, excipient and supplement in food, cosmetics, medicine and feed.

Example 11 Conversion of Starch Hydrolysates by Recombinant Enzymes

Tapioca starch was dissolved in water to a concentration of 34 w / w%, and 0.1 w / w% calcium carbonate was added and mixed. To the suspension, α-amylase product "TERMAMYL 60L" (sold by Novo Nordisk Bioindustri A / S, Denmark) was added 0.2 w / w% per 1 g of starch on a dry solids basis, followed by enzymatic reaction at 95 ° C for 15 minutes. Was liquefied. The liquefied starch was autoclaved at 120 ° C. for 10 minutes to inactivate the remaining enzymes, quench to 55 ° C. to adjust pH to 5.2, and dry the α-amylase product “α-amylase 2A” (sold by Ueda Chemical of Japan). As a standard, 10 units per 1 g of starch and 500 units of isoamylase (available from Hayashibara Biochemical Laboratories, Japan) were added and mixed, followed by enzymatic reaction at 55 ° C. for 20 hours, maltotriose, maltotetraose, Reducing starch saccharides such as maltopentaose and maltohexaose contained about 60 w / w% on a dry solids basis to obtain a mixture of DE about 29. The mixture was autoclaved at 120 ° C. for 10 minutes to inactivate the remaining enzymes, quench to 45 ° C. to adjust pH to 6.5, and then adjust the recombinant enzyme prepared by the method of Example 6 to 1 g per starch on a dry solids basis. Units were added and mixed, followed by enzymatic reaction at 45 ° C for 64 hours. The obtained reaction mixture was heated at 95 ° C. for 10 minutes to deactivate the remaining enzyme, cool down, filter, decolorize with activated carbon in a conventional manner, desalter and purify with ion exchanger, and then concentrated to a dry solid content of about 70 w / w Syrup-like products in% concentration were obtained with a yield of about 90% on a dry solids basis relative to the raw starch.

As a result of analyzing the syrup phase product by the method of Experiment 2-1, DE was 15.8 and 5.8 w / w% α-glucosyl trehalose, 8,5 w / w% α-maltosyl trehal based on dry solids Loose, 13,1 w / w% α-maltotriosyl trehalose, 18.9 w / w% α-maltotetraosyl trehalose and 3.6 w / w% α-maltopentaosil trehalose It was found to be the main component, and it was confirmed that most of the reducing starch sugars contained therein were converted to their respective non-reducing sugars. This product has mild to moderate sweetness, moderate viscosity and moisturizing properties, so it can be used satisfactorily as a sweetener, taste improver, quality improver, stabilizer, filler, excipient and supplement in food, cosmetics and medicine. In addition, this product contains a relatively large amount of non-reducing sugars and can be used as an intermediate for preparing trehalose.

Example 12 Conversion of Hydrolysates by Recombinant Enzymes

As in Example 8, maltotetraose producing enzyme and α-amylase were successively acted on liquefied potato starch to reduce starch saccharides such as maltotriose, maltotetraose and maltopentaose having a glucose degree of polymerization of 3 or more based on dry solids. A mixture containing about 50 w / w% was prepared. The reaction mixture was autoclaved at 120 ° C. for 10 minutes to inactivate the remaining enzymes, quench to 45 ° C. to adjust pH to 6.5, and then adjust the recombinant enzyme prepared in Example 6 to 1 g per starch on a dry solids basis. 2 units were added and mixed, followed by enzymatic reaction at 45 ° C for 64 hours. The obtained reaction mixture was heated at 95 ° C. for 10 minutes to deactivate the remaining enzyme, cool and filter, and then the filtrate was decolorized with activated charcoal, desalted, purified with ion exchanger, and concentrated to about 70 w / w% syrup. The phase product was obtained in a yield of about 90 w / w% based on dry solids relative to the raw starch.

As a result of analyzing the syrup phase product by the method of Experiment 2-1, DE was 10.3 and 3.6 w / w% α-glucosyl trehalose, 44.0 w / w% α-maltosyl trehalose based on dry solids And 1.0 w / w% α-maltotriosyl trehalose as the main component, and it was confirmed that most of the reducing starch sugars contained in the syrup-like product were converted to their respective non-reducing sugars.

This product has mild to moderate sweetness, moderate viscosity and moisturizing properties, so it can be used satisfactorily as a sweetener, taste improver, quality improver, stabilizer, filler, excipient and auxiliary agent in food, cosmetics and medicines. In addition, this product contains a relatively large amount of non-reducing sugars and can be used as an intermediate for preparing trehalose.

As described above, the present invention is based on the discovery of a novel enzyme that produces a non-reducing sugar having a trehalose structure as a terminal unit from a reducing sugar having a glucose degree of polymerization of 3 or more. The present invention seeks to produce this enzyme in a relatively high yield on a relatively large scale by recombinant DNA techniques. By the conversion method using the recombinant enzyme of the present invention, reducing starch sugars have a mild high-quality sweetness, moderate viscosity and moisturizing properties, but have no reducing residues in the molecule and sweeten the sample without fear of unnecessary coloring or deterioration. It effectively converts each corresponding non-reducing sugar into which it can be endowed. Moreover, the recombinant enzyme of the present invention can be used in the preparation of non-reducing sugars and trehalose having a trehalose structure as a terminal unit that can be used in foods without fear of side effects since the entire amino acid sequence has been confirmed.

Therefore, this invention is an important invention which can show the outstanding effect | action and effect as mentioned above, and can make a big contribution to industry.

1 is a graph showing the optimum temperature of enzyme M-11.

2 is a graph showing the optimal temperature of enzyme Q36.

3 is a graph showing the optimal pH of enzyme M-11.

4 is a graph showing the optimal pH of enzyme Q36.

5 is a graph showing the thermal stability of enzyme M-11.

6 is a graph showing the thermal stability of enzyme Q36.

7 is a graph showing pH stability of enzyme M-11.

8 is a graph showing pH stability of enzyme Q36.

9 is a restriction diagram of the recombinant DNA pBMT7 according to the present invention, in which the bold line indicates DNA encoding the enzyme M-11.

Fig. 10 is a restriction diagram of recombinant DNA pBQT13 according to the present invention, in which the part shown in bold lines is DNA encoding the enzyme Q36.

Claims (27)

DNA encoding an enzyme that produces a non-reducing sugar having a trehalose structure as a terminal unit from a reducing starch sugar having a glucose degree of polymerization of 3 or more, wherein the DNA is genus Rizobium, Ardrobacter, Bre It is derived from a microorganism selected from the group consisting of the genus Nonbacterium, Flavobacterium, Microcosus, Kurtobacterium, Mycobacterium, and Terrabacter, and also the following SEQ ID NO: 1 and SEQ ID NO : Has base sequences starting at the 5 'end as shown in 3, and (b) the enzyme consists of amino acid sequences starting at the N end as shown in SEQ ID NO: 2 and SEQ ID NO: 4. DNA having an amino acid sequence selected from the group and having the following physical and chemical properties. Physicochemical Properties of Enzymes (1) molecular weight 76,000 to 87,000 Daltons in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). (2) isoelectric point (pI) 3.6 to 4.6 at isoelectric point electrophoresis The amino acid of claim 1, wherein at least one base in SEQ ID NO: 1 and SEQ ID NO: 3 as defined in claim 1 is selected from amino acids in SEQ ID NO: 2 and SEQ ID NO: 4 as defined in claim 1. DNA substituted with another base by degeneracy of the genetic code without altering the sequence. The DNA of claim 1 having the nucleotide sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 11. A replicable DNA containing the DNA of claim 1 and a vector capable of replicating itself. The method of claim 4, wherein at least one base in SEQ ID NO: 1 and SEQ ID NO: 3 as defined in claim 1 corresponds to each of SEQ ID NO: 2 and SEQ ID NO: 4 as defined in claim 1. Replicable DNA substituted with another base by degeneracy of the genetic code without changing one amino acid sequence. The replicable DNA of claim 4 having a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NO: 10 and SEQ ID NO: 11 as defined in claim 3. The replicable DNA of claim 4, wherein said self-replicating vector is a plasmid vector Bluescript II SK (+). A bacterium obtained by introducing a vector capable of self-replicating into a suitable host and a DNA containing the DNA of claim 1. The method according to claim 8, wherein at least one base of SEQ ID NO: 1 and SEQ ID NO: 3 as defined in claim 1 corresponds to each of SEQ ID NO: 2 and SEQ ID NO: 4 as defined in claim 1. Bacteria substituted by another base by degeneracy of the genetic code without altering one amino acid sequence. The bacterium of claim 8, wherein the DNA of claim 1 is selected from the group consisting of the nucleotide sequences in SEQ ID NO: 10 and SEQ ID NO: 11 as defined in claim 6. The bacterium of claim 8, wherein the self-replicating vector is a plasmid vector Bluescript II SK (+). The bacterium of claim 8, wherein said host is a microorganism of Escherichia coli species. A recombinant enzyme obtained from a bacterium of claim 8 which produces a non-reducing sugar having a trehalose structure as a terminal unit from a reducing starch sugar having a glucose degree of polymerization of 3 or more, wherein (a) the DNA is a genus of rizobium, Ardrobacter Is derived from a microorganism selected from the group consisting of genus, genus Brevibacterium, genus Flavobacterium, genus Microcus, genus Courtobacterium, mycobacterium and Terrabacter, and (b) the enzyme is SEQ ID NO. : A recombinant enzyme having an amino acid sequence selected from the group consisting of amino acid sequences starting at the N terminus as shown in SEQ ID NO: 2 and SEQ ID NO: 4, and also having the following physical and chemical properties. Physicochemical Properties of Enzymes (1) molecular weight 76,000 to 87,000 Daltons in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), (2) isoelectric point (pI) 3.6 to 4.6 at isoelectric point electrophoresis A method for producing the recombinant enzyme of claim 13, comprising culturing the bacteria of claim 8 capable of producing the recombinant enzyme of claim 13 in a nutrient medium, and harvesting the resulting recombinant enzyme from the obtained culture solution. 15. The bacterium of claim 14, wherein the bacterium is capable of self replication with the DNA of claim 1 encoding an enzyme that produces a non-reducing sugar having a trehalose structure as a terminal unit from a reducing starch sugar having a glucose polymerization degree of 3 or more. A method for producing a product, which is obtained by introducing a DNA having a vector into a suitable host. 16. The method of claim 15, wherein the DNA encoding the enzyme comprises a base sequence starting at the 5 'end as in SEQ ID NO: l and SEQ ID NO: 3 as defined in claim 1, Base complementary to SEQ ID NO: 1 and SEQ ID NO: 3 obtained by substituting one or more bases in SEQ ID NO: 1 and SEQ ID NO: 3 with another base without substantially changing the intrinsic activity of the enzyme And a base sequence selected from the group consisting of arrays. The method of claim 16, wherein the DNA encoding the enzyme is one or more bases of the genetic code without changing the respective amino acid sequence of SEQ ID NO: 2 and SEQ ID NO: 4 defined in claim 1 And a base sequence selected from the group consisting of the base sequences of SEQ ID NO: 1 and SEQ ID NO: 3 as defined in claim 1, which are substituted with other bases by derivatization. 15. The method according to claim 14, wherein the enzyme is encoded by DNA having a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NO: 10 and SEQ ID NO: 11 as defined in claim 3. Manufacturing method. The method of claim 14, wherein the bacterium is produced using a microorganism of Escherichia coli species as a host. The method of claim 14, wherein the bacterium is prepared using the plasmid vector Bluescript II SK (+) as a vector capable of self replication. The method according to claim 14, wherein the bacteria are inoculated in a liquid medium having a pH of 2 to 8 and incubated for 1 to 6 days at a temperature of 25 to 65 ° C. The method according to claim 14, wherein the enzyme in the culture medium is subjected to centrifugation, filtration, concentration, salting out, dialysis, ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, affinity chromatography, gel electrophoresis and isoelectric electrophoresis. A production method characterized by harvesting by one or more methods selected from the group. An improvement of the method for converting reducing starch sugars, wherein the improved product is obtained by culturing the bacteria of claim 8 capable of producing the enzyme of claim 25, and extracting the enzyme produced from the culture medium to prepare the enzyme of claim 13, and the degree of glucose polymerization is 3 A method of converting a reducing starch sugar, comprising the step of reacting the above-described reducing starch sugar with the recombinant enzyme to produce a non-reducing sugar having a trehalose structure as a terminal unit. The conversion method according to claim 23, wherein the reducing starch sugar is one selected from the group consisting of starch hydrolysate and starch material treated or not treated with amylase. 24. The method according to claim 23, wherein the reducing starch sugar is one selected from the group consisting of maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, and mixtures thereof. 24. The process of claim 23, wherein the reducing starch sugar is in the form of a solution having a concentration of 50 w / v% or less, and the process steps are carried out at a temperature of 40 to 55 ° C and at a pH of 5 to 10. 24. The method of claim 23, wherein the non-reducing sugar is α-glucosyl trehalose, α-maltosyl trehalose, α-maltotriosyl trehalose, α-maltotetraosyl trehalose, α -A conversion method characterized in that it is one kind selected from the group consisting of maltopentaosyl trehalose and mixtures thereof.